Laboratory Methods in Enzymology: Protein Part B

Contributors

Miscellaneous

Preface

Section I: Protein Protocols/Protein In Vitro Translation

Chapter One: In Vitro Synthesis of Proteins in Bacterial Extracts

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Grow and harvest E. coli for the S30 extract

6 Step 2 Preparation of the S30 extract

7 Step 3 Optimization of the coupled transcription and translation reaction

Chapter Two: Preparation of a Saccharomyces cerevisiae Cell-Free Extract for In Vitro Translation

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Preparation of Yeast Cell-Free Extract

6 Step 2 Cell-Free Translation

Section II: Protein Protocols/Protein In Vivo Binding Assays

Chapter Three: Yeast Two-Hybrid Screen

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Small-Scale Transformation of Yeast with pDBLeu-X

6 Step 2 Two-Hybrid Screen

7 Step 3 Confirmation of Positive Interactors

8 Step 4 Plasmid Rescue from Yeast

9 Step 5 Electroporation of E. coli with Yeast DNA and Identification of Positive Interactors

10 Step 6 Back-Transformation of Yeast and Further Confirmation of Interactions

Chapter Four: UV Cross-Linking of Interacting RNA and Protein in Cultured Cells

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 UV Cross-Link RNA–Protein Complexes

6 Step 2 SDS Lysis of Cells

7 Step 3 Immunoprecipitation

8 Step 4 Proteinase K Treatment of RNA Samples

9 Step 5 RNA Analysis

10 Step 6 Protein Analysis

Chapter Five: Analysis of RNA–Protein Interactions by Cell Mixing

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Cell Mixing

6 Step 2 Cell Lysis

7 Step 3 Immunoprecipitation

8 Step 4 Proteinase K Treatment of RNA Samples

9 Step 5 Northern and Western Blot Analysis

Chapter Six: General Protein–Protein Cross-Linking

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Calculate the Amount of BS3 to Use

6 Step 2 Protein Cross-Linking

Chapter Seven: Chromatin Immunoprecipitation and Multiplex Sequencing (ChIP-Seq) to Identify Global Transcription Factor Binding Sites in the Nematode Caenorhabditis Elegans

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Preparation of Extract from Formaldehyde-Fixed Caenorhabditis elegans Embryos and Larvae

6 Step 2 Washes and Collection of the Immunocomplexes and ChIP DNA Purification

7 Step 3 Library Preparation for Multiplex Sequencing Using the Illumina Genome Analyzer

Acknowledgments

Chapter Eight: PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 UV Crosslinking of 4-Thiouridine-Labeled Cells (Day 1)

6 Step 2 Preparation of Cell Lysate for Immunoprecipitation (Day 2)

7 Step 3 Preparation of the Magnetic Beads (Day 2)

8 Step 4 Immunoprecipitation and Second RNase T1 Treatment (Day 2)

9 Step 5 Dephosphorylation and Radiolabeling RNA Segments Crosslinked to Immunoprecipitated Proteins (Day 2)

10 Step 6 SDS-PAGE and Electroelution of Cross-Linked RNA-Protein Complexes from Gel Slices (Days 2 and 3)

11 Step 7 Proteinase K Digestion (Day 3)

12 Step 8 3′-Adapter Ligation for cDNA Library Preparation (Day 3 overnight, day 4, beginning of day 5)

13 Step 9 5′-Adapter Ligation for cDNA Library Preparation (Day 5, beginning of day 6)

14 Step 10 cDNA Library Preparation/Reverse Transcription (Day 6)

15 Step 11 PCR Amplification of cDNA Library & Sample Preparation for Sequencing (Day 6)

16 Step 12 Determination of Incorporation Levels of 4SU into Total RNA

Chapter Nine: Determining the RNA Specificity and Targets of RNA-Binding Proteins using a Three-Hybrid System

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency

6 Step 2 Determine 3-AT Concentration to be Used in Selection

7 Step 3 Introduce the Hybrid RNA Library

8 Step 4 Assay β-Galactosidase Activity

9 Step 5 Cure the RNA Plasmid and Test Positives for Protein Dependence

10 Step 6 Isolate Plasmids for Autoactivation Test and Sequencing

11 Step 7 Determine Binding Specificity Using Mutant and Control Proteins

12 Step 8 Functional Tests or Additional Screens

Chapter Ten: Dissecting a Known RNA–Protein Interaction using a Yeast Three-Hybrid System

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1A Assaying Interactions: Qualitative Filter Assay for β-Galactosidase Activity

6 Step 1B Assaying Interactions: Quantitative Solution Assay for β-Galactosidase Activity

7 Step 1C Assaying Interactions: 3-Aminotriazole (3-AT) Resistance Assay

Chapter Eleven: Identifying Proteins that Bind a Known RNA Sequence Using the Yeast Three-Hybrid System

1 Theory

2 Equipment

3 Materials

4 Protocol

5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency

6 Step 2 Determine 3-AT Concentration to be Used in Selection

7 Step 3 Introduce the cDNA Library

8 Step 4 Eliminate RNA-Independent False Positives by Colony Color

9 Step 5 Assay β-Galactosidase Activity

10 Step 6 Cure the RNA Plasmid and Test Positives for RNA Dependence

11 Step 7 Isolate Plasmids for Autoactivation Test and Sequencing

12 Step 8 Determine Binding Specificity Using Mutant and Control RNAs

13 Step 9 Functional Tests or Additional Screens

Author Index

Subject Index